Stem cells are some of the longest-lived cells in our bodies, with a lifespan that can exceed dozens of years. They are critical to our long-term health, and age-related dysfunction in stem cells is linked to cancer, diabetes and organ failure. The objective of this proposal is to determine how stem cells and other mitotically-active cells maintain cell health and avoid premature aging. The lifespan of stem cells includes periods of proliferation and quiescence (a reversible, sustainable cell state of non-proliferation). When proliferating, part of the stem cell population must replace cells that differentiate or die. The overarching hypothesis guiding this proposal is that cells utilize quiescence to reduce reactive oxygen species (ROS) produced during aerobic respiration in mitochondria to preserve their replicative lifespan. The aims of this proposal will elucidate (1) if quiescence can extend the lifespan of cells by reducing intracellular ROS, and (2) if mitochondrial dynamics (fusion, fission and distribution at division) are managed differently by cells during quiescence and proliferation to achieve full replicative lifespan. This research introduces an innovative, high-throughput microfluidic device for studying replicative lifespan in the fission yeast Schizosaccharomyces pombe, a classic model for mitotically active, symmetrically dividing cells. The fission yeast lifespan microdissector (FYLM) is a microfluidic device that captures and immobilizes hundreds of fission yeast cells over their entire replicative lifespan, while they are kept in a constant, controllable environment and observed with subcellular resolution. (1) Using the FYLM and fluorescent ROS reporters of ROS, I will determine the interplay between replicative lifespan and proliferation after quiescence in S. pombe. Specifically, we will test the hypothesis that cell reduce intracellular ROS levels during quiescence to extend their replicative lifespan. (2) Next, I will directly observe the distribution and dynamics of aged mitochondria over the replicate lifespan of individual S. pombe cells. Finally, mitochondrial fusion and fission will be regulated n engineered strains to determine whether the overall turnover rate of mitochondria or the ratio of fusion to fission are critical to the cell health during proliferation and quiescence. I hypothesiz that symmetrically dividing cells equally divide mitochondria over their entire replicative lifespa, instead depending on rapid mitochondrial turnover to remove dysfunctional mitochondrial, and that during quiescence, precise balance of fusion and fission maintains mitochondrial homeostasis. Completion of these aims will delineate how symmetrical division and quiescence support longevity in a eukaryotic model for long- lived, mitotically active cells. Ultimately, thes studies will shed light on how stem cells and other long-lived eukaryotic cells are able to maintain cellular health.